Tularemia Pathogenesis Section, Laboratory of Intracellular Parasites, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA.
Autophagy. 2012 Sep;8(9):1342-56. doi: 10.4161/auto.20808. Epub 2012 Aug 6.
Cytosolic bacterial pathogens must evade intracellular innate immune recognition and clearance systems such as autophagy to ensure their survival and proliferation. The intracellular cycle of the bacterium Francisella tularensis is characterized by rapid phagosomal escape followed by extensive proliferation in the macrophage cytoplasm. Cytosolic replication, but not phagosomal escape, requires the locus FTT0369c, which encodes the dipA gene (deficient in intracellular replication A). Here, we show that a replication-deficient, ∆dipA mutant of the prototypical SchuS4 strain is eventually captured from the cytosol of murine and human macrophages into double-membrane vacuoles displaying the late endosomal marker, LAMP1, and the autophagy-associated protein, LC3, coinciding with a reduction in viable intracellular bacteria. Capture of SchuS4ΔdipA was not dipA-specific as other replication-deficient bacteria, such as chloramphenicol-treated SchuS4 and a purine auxotroph mutant SchuS4ΔpurMCD, were similarly targeted to autophagic vacuoles. Vacuoles containing replication-deficient bacteria were labeled with ubiquitin and the autophagy receptors SQSTM1/p62 and NBR1, and their formation was decreased in macrophages from either ATG5-, LC3B- or SQSTM1-deficient mice, indicating recognition by the ubiquitin-SQSTM1-LC3 pathway. While a fraction of both the wild-type and the replication-impaired strains were ubiquitinated and recruited SQSTM1, only the replication-defective strains progressed to autophagic capture, suggesting that wild-type Francisella interferes with the autophagic cascade. Survival of replication-deficient strains was not restored in autophagy-deficient macrophages, as these bacteria died in the cytosol prior to autophagic capture. Collectively, our results demonstrate that replication-impaired strains of Francisella are cleared by autophagy, while replication-competent bacteria seem to interfere with autophagic recognition, therefore ensuring survival and proliferation.
胞内细菌病原体必须逃避细胞内先天免疫识别和清除系统，如自噬，以确保其存活和增殖。细菌弗朗西斯菌的细胞内周期的特点是快速吞噬体逃逸，然后在巨噬细胞质中广泛增殖。细胞质复制，但不是吞噬体逃逸，需要FTT0369c 基因座，该基因座编码 dipA 基因（细胞内复制 A 缺陷）。在这里，我们表明，一种复制缺陷的、∆dipA 突变体的原型 SchuS4 株最终从鼠和人巨噬细胞的细胞质中捕获到双层膜囊泡，该囊泡显示晚期内体标记物 LAMP1 和自噬相关蛋白 LC3，同时活细胞内细菌数量减少。SchuS4ΔdipA 的捕获不是 dipA 特异性的，因为其他复制缺陷细菌，如氯霉素处理的 SchuS4 和嘌呤营养缺陷突变体 SchuS4ΔpurMCD，也被靶向到自噬小泡。含有复制缺陷细菌的小泡被泛素和自噬受体 SQSTM1/p62 和 NBR1 标记，并且它们的形成在来自 ATG5-、LC3B- 或 SQSTM1 缺陷型小鼠的巨噬细胞中减少，表明通过泛素-SQSTM1-LC3 途径识别。虽然野生型和复制受损株的一部分都被泛素化并募集 SQSTM1，但只有复制缺陷株进展到自噬捕获，表明野生型弗朗西斯菌干扰自噬级联。自噬缺陷型巨噬细胞中复制缺陷株的存活没有恢复，因为这些细菌在自噬捕获之前在细胞质中死亡。总之，我们的结果表明，复制受损的弗朗西斯菌菌株通过自噬清除，而复制能力的细菌似乎干扰自噬识别，因此确保存活和增殖。
